Regularly arrayed surface layers have been observed on a wide variety of bacterial species. The tetragonally arrayed layer (T-layer) of Bacillus sphaericus forms the outermost structure of this bacterium and is composed of identical protein subunits with a molecular weight of 160,000 daltons. The T-layer of B. sphaericus can be solubilized by acid and reassembled at pH 7.0 to yield a tetragonally arrayed structure identical to that seen on whole cells. The assembly and mechanism of growth of T-layer on the surface of B. sphaericus will be studied in these proposed experiments. The sites of integration of new T-layer subunits will be located using two markers specific for T-layer protein. These are specific antisera directed against T-layer and bacteriophage M, which attaches only to the T-layer structure. The influence of peptidoglycan on in vivo assembly of T-layer will be studied using sphaeroplasts and cells treated with known inhibitors of peptidoglycan synthesis. Previous published data suggested that T-layer is essential for the viability or growth of B. sphaericus. In additional studies, the function of T-layer protein will be investigated by isolation of temperature sensitive mutants which fail to deposit T-layer subunits on the cell surface during growth at the restrictive temperature. Bacteriophage M will be used to select for mutants devoid of T-layer. Cells lacking the T-layer will be examined for aberrations in morphology, divison, and synthesis of other cell wall polymers.